无涡旋和离心的 HIV-1 RNA 提取。
Vortex- and Centrifugation-Free Extraction of HIV-1 RNA.
机构信息
Center for Biomedical Engineering, School of Engineering, Brown University, 182 Hope Street, Providence, RI, 02912, USA.
PerkinElmer, 940 Winter Street, Waltham, MA, 02451, USA.
出版信息
Mol Diagn Ther. 2019 Jun;23(3):419-427. doi: 10.1007/s40291-019-00394-1.
BACKGROUND AND OBJECTIVE
HIV viral load measurements play a critical role in monitoring disease progression in those who are on antiretroviral treatment. In order to obtain an accurate measurement, rapid sample preparation techniques are required. There is an unmet need for HIV extraction instruments in resource-limited settings, where HIV prevalence is high. Therefore, the objective of our study was to develop a three-dimensional (3D) microfluidic system to extract HIV-1 RNA with minimal electricity and without complex laboratory instruments.
METHODS
A 3D microfluidic system was designed in which magnetic beads bound with nucleic acids move through immiscible oil-water interfaces to separate HIV-1 RNA from the sample. Polymerase chain reaction (PCR) amplification was used to quantify the total amount of HIV-1 RNA extracted as we optimized the system through chip design, bead type, carry-over volume, carrier RNA concentration, and elution buffer temperature. Additionally, the extraction efficiency of the 3D microfluidic system was evaluated by comparing with a Qiagen EZ1 Advanced XL instrument using 20 HIV-1-positive plasma samples.
RESULTS
Our method has near-perfect (100%) extraction efficiency in spiked serum samples with as little as 50 copies/mL starting sample. Furthermore, we report carry-over volumes of 0.31% ± 0.006% of total sample volume. Using the EZ1 Advanced XL as a gold standard, the average percentage HIV-1 RNA extracted using the microchip was observed to be 65.4% ± 24.6%.
CONCLUSIONS
From a clinical perspective, the success of our method opens up its possible use in diagnostic tests for HIV in the remote areas where access to vortexes and centrifuges is not available. Here we present a proof-of-concept device which, with further development, could be used for sample preparation at the point of care.
背景和目的
HIV 病毒载量测量在接受抗逆转录病毒治疗的患者的疾病进展监测中起着至关重要的作用。为了获得准确的测量结果，需要快速的样品制备技术。在资源有限的环境中，HIV 提取仪器的需求尚未得到满足，而这些地区的 HIV 流行率较高。因此，我们的研究目标是开发一种三维（3D）微流控系统，以最小的电量和无需复杂的实验室仪器提取 HIV-1 RNA。
方法
设计了一种 3D 微流控系统，其中与核酸结合的磁性珠通过不混溶的油水界面移动，以将 HIV-1 RNA 从样品中分离出来。通过芯片设计、珠类型、携带体积、载体 RNA 浓度和洗脱缓冲液温度等方面对系统进行优化，使用聚合酶链反应（PCR）扩增来定量提取的 HIV-1 RNA 的总量。此外，使用 20 份 HIV-1 阳性血浆样本，通过与 Qiagen EZ1 Advanced XL 仪器进行比较，评估了 3D 微流控系统的提取效率。
结果
我们的方法在起始样本为 50 拷贝/mL 的模拟血清样本中具有近乎完美的（100%）提取效率。此外，我们报告的携带体积为总样品体积的 0.31%±0.006%。使用 EZ1 Advanced XL 作为金标准，使用微芯片提取的 HIV-1 RNA 的平均百分比为 65.4%±24.6%。
结论
从临床角度来看，我们的方法的成功为在无法获得漩涡和离心机的偏远地区进行 HIV 诊断测试开辟了可能。在这里，我们提出了一种概念验证设备，如果进一步开发，它可以用于即时护理的样品制备。
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